Aconitase (Beasley)



Aconitase (ACO) is an enzymatic domain that catalyzes the reversible isomerization of citrate and L-isocitrate using cis-aconitate as an intermediate:
 * citrate⇌aconitate⇌L-isocitrate

This reaction is part of the citric acid cycle, which is also known as the tricarboxylic acid cycle (TCA cycle) or the Krebs cycle. The images at the left and at the right correspond to one representative Aconitase, i.e. the crystal structure of Bovine Aconitase (1amj). In most organisms, there is a cytosolic enzyme with an ACO domain (cAc), and in eukaryotes, there is also a mitochondrial aconitase (mAc). Plants developed even more copies in mitochondria. Aconitase contains a [4Fe-4S] iron-sulfur cluster which converts to [3Fe-4S] when the enzyme is inactive. In humans, two types of ACO are expressed: the soluble ACO1 and the mitochondrial ACO2. Aconitase from pig (PDB 7acn) is a single polypeptide (Mr 83kD) that catalyzes the reversible isomerization of citrate and L-isocitrate. It is the second enzyme in the Citric acid cycle, which is a series of enzyme-catalysed chemical reactions that is crucial to aerobic cellular respiration and the production of ATP.

Structure
 The secondary structure consists of numerous alternating alpha helices and beta sheets (SCOP classification α/β alternating). The tertiary structure is somewhat bilobed with the active site in the middle, and, since there is only one subunit, there is no quaternary structure. Aconitase consists of four domains, three of which are tightly packed while the fourth is more flexible. Aconitase contains a 4Fe-4S iron-sulfur cluster. Iron-sulfur clusters occur as the prosthetic groups of iron-sulfur proteins which are also called nonheme iron proteins. [4Fe-4S] clusters are coordinated to four protein Cys sulfhydryl groups with the Fe atoms in the cluster coordinated by four S atoms, which are tetrahedrally disposed around the Fe. This iron sulfur cluster does not participate in redox as most do, but holds the OH group of citrate to facilitate its elimination. It is at this [4Fe-4S] site that catalysis occurs and citrate or isocitrate is bound. The rest of the active site is made up of residues Gln72, Asp100, His101, Asp165, Ser166, His167, His147, Glu262, Asn258, Cys358,Cys424, Cys358, Cys421, Asn446, Arg447, Arg452, Asp568, Ser642, Ser643, Arg644, Arg580.

Catalytic mechanism of mitochondrial ACO
Both mAc and cAc are quite similar in their ACO function. Studies, however, concentrate on the mitochondrial ACO. ACO is an excellent system for understanding the role of iron-sulfur-clusters in catalysis. The [4Fe-4S]cofactor is held in place by three sulfur atoms belonging to the cysteins-358, -421, and -424 which are bound to three of the four cluster iron atoms (Cys 421 and Cys 424 are connected by Gly 422 and Pro 423 in this image). On activation of the enzyme, a fourth iron atom is included in the cluster together with a water molecule.This Fe4 is free to bind one, two, or three partners, in this reaction always oxygen atoms belonging to other molecules.

Substrate-free aconitase contains a [4Fe-4S]2+ cluster with hydroxyl bound to one of the Fe. Upon binding of substrate the bound hydroxyl is protonated. A hydrogen bond from His101 to the isocitrate hydroxyl is donated to form water. Alternatively, the proton could be donated by His167 as this histidine is hydrogen bonded to a H2O molecule. His167 is also hydrogen bonded to the bound H2O in the [4Fe-4S] cluster. Both His101 and His167 are paired with carboxylates (Asp100 and Glu262, respectively) and are likely to be protonated. The conformational change associated with substrate binding reorients the cluster. The residue which removes a proton from citrate or isocitrate is Ser642. This causes the cis-Aconitate intermediate (seen below), which consists of a double bond, which is a direct result of the deprotonation. Then, there is a rehydration of the double bond of cis-aconitate to form isocitrate (if the original substrate was citrate. Even though addition of water across the double bond of cis-Aconitate could yield four stereoisomers, aconitase catalyzes the stereospecific addition of the hydroxyl group and hydrogen to produce only one isocitrate stereoisomer. To better understand this, consider this process as stages, seen below.

Stage 1: Dehydration
First, dehydration of citrate causes a proton and OH group to be removed from only the 'lower arm'. This forms a cis-Aconitate intermediate.

Stage 2: Rehydration
The second main stage of the reaction is the rehydration of the cis-Aconitate intermediate. This forms isocitrate. It is catalyzed in a stereospecific way such that only one isocitrate stereoisomer is formed.

Thus, the overall reaction that aconitase catalyzes is: Citrate⇌Aconitate⇌Isocitrate, as seen below (in the forward direction):

Regulation
Aconitase can be inhibited or activated to increase or decrease the ability to catalyze the reaction of citrate to isocitrate. The activity of aconitase can be reduced when one Fe is lost from the cluster. This lowers the activity over 100-fold, but then can regain full activity by adding another Fe from solution. Aconitase is also strongly inhibited by nitro analogs

The Citric Acid Cycle works in such a way that the product of one reaction becomes the reactant of another, with different enzymes catalyzing each reaction. Aconitase is one such enzyme. Some of these enzymes are tightly regulated, either activated or inhibited, by the concentration of reactant, product, ATP or NADH, and thus are rate-determining. Aconitase is not one of the three rate-determining enzymes of the Citric Acid Cycle as its ΔG is not negative (ΔG°′≈5 kJ/mol and ΔG≈0 kJ/mol). Aconitase functions close to equilibrium and the rate of citrate consumption depends on the activity of NAD+-dependent isocitrate dehydrogenase, which is one of the three rate-determining enyzmes. Isocitrate dehydrogenase uses the product of the reaction aconitase catalyzes. Both Citrate synthase and Isocitrate dehydogenase are inhibited by NADH concentration, but aconitase itself is not. If NADH concentration were high, then NAD+-dependent isocitrate dehydrogenase wouldn’t catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate. This would lead to a buildup of isocitrate and aconitase would catalyze the reversible isomerization of citrate and isocitrate to provide more reactant since there was an increase in product. A buildup of citrate would inhibit the enzyme of the previous step, citrate synthase. An illustration of the relationship that these three enzymes have on each other is seen below, with the boxes representing the enzymes that catalyze each reaction. This is a common example of how the Citric Acid Cycle works in order to produce ATP without wasting resources. Similar inhibition/activation of enzymes occurs based on concentrations of ATP, NADH, Calcium, CoA, and others.

Cytosolic aconitase and its other function
A specialty of cAc is that in mammals it has developed a <scene name='Aconitase/2ipy-total/2'>second function as inhibitor of <scene name='Aconitase/2ipy-rna/1'>those mRNA that carry an <scene name='Aconitase/2ipy-rna-ire/1'>iron-responsive element (IRE). Therefore, the cytosolic aconitase,cAc,is named IREBP for IRE-binding protein when this function is talked about. Iron regulatory protein-1 (IRP-1), is both a cytoplasmic aconitase and a regulatory RNA-binding protein. These findings suggests a novel role for Fe-S clusters as post-translational regulatory switches. Only one of the two functions (cytoplasmic aconitase or regulatory RNA-binding protein) is active, depending on whether <scene name='Aconitase/2b3x-cluster/1'>the [4Fe-4S] cofactor is present in the molecule. The [4Fe-4S] cofactor is essential for <scene name='Aconitase/2b3x-total/1'>the ACO function. In the presence of iron, a [4Fe-4S] cluster assembles in IRP-1, converting it from an RNA-binding protein into a cytosolic aconitase. You can see, by <scene name='Aconitase/Morph/2'>looking at the morph, how much the enzyme structure differs between those two functions.

ACO
1b0k – pACO (mutant) – pig

5acn – pACO+Fe3S4

6acn - pACO+Fe4S4

1amj, 1nit – cACO - cow

ACO+citrate
1c96 - pACO (mutant)+citrate

1b0m - pACO (mutant)+fluorocitrate

ACO+aconitate
1fgh – cACO+4-hydroxy-aconitate

1aco – cACO+transaconitate

1nis - cACO+transaconitate+nitrocitrate

ACO+isocitrate
7acn - pACO +isocitrate

1c97, 1b0j - pACO (mutant)+isocitrate

1ami, 8acn – cACO+isocitrate

ACO1
2b3x, 2b3y – hACO1 – human

2ipy – rACO1 (mutant)+ferritin H IRE-RNA – rabbit

ACO2
1l5j – ACO2 – Escherichia coli

Literature

 * M. Claire Kennedy and Helmut Beinert: IX.4. Aconitase. in Ivano Bertini, Harry B. Gray, Edward I. Stiefel, Joan Selverstone Valentine (eds.): Biological Inorganic Chemistry: Structure and Reactivity. University Science Books, Herndon 2006. ISBN 1891389432 pp.209--

Additional Resources
For additional information, see: Carbohydrate Metabolism